GLUD1基因对人牙髓干细胞的体外增殖、分化和矿化的影响

doi:10.19439/j.sjos.2017.04.002

上海口腔医学 ›› 2017, Vol. 26 ›› Issue (4): 358-362.doi: 10.19439/j.sjos.2017.04.002

GLUD1基因对人牙髓干细胞的体外增殖、分化和矿化的影响

卢皙, 刘上峰, 赵守亮

复旦大学附属华山医院口腔科,上海 200040

收稿日期:2017-03-21 修回日期:2017-05-03 出版日期:2017-08-25 发布日期:2017-09-01
通讯作者: 赵守亮,E-mail:slzhao@fudan.edu.cn
作者简介:卢皙（1990-）,女,硕士研究生,E-mail:imlucy@aliyun.com
基金资助:
国家自然科学基金（81170951,81570965,81070825）

Effect of GLUD1 on proliferation, osteogenic differentiation and mineralization of human dental pulp stem cells

LU Xi, LIU Shang-feng, ZHAO Shou-liang

Department of Stomatology, Huashan Hospital, Fudan University. Shanghai 200040, China

Received:2017-03-21 Revised:2017-05-03 Online:2017-08-25 Published:2017-09-01

摘要/Abstract

摘要： 目的探索谷氨酸脱氢酶1（Glutamate dehydrogenase 1,GLUD1）基因在人牙髓干细胞（human dental pulp stem cells,hPDCSs）体外增殖、成骨分化和矿化中的作用。方法体外分离培养hPDCSs,利用慢病毒感染方式沉默GLUD1基因的表达,分别采用RT-PCR和Western免疫印迹检测mRNA及蛋白水平的沉默效率。采用CCK8法检测细胞的体外增殖水平。对hDPSC进行体外成骨诱导分化培养,采用茜素红染色法检测矿化结节的形成,利用RT-PCR和免疫荧光染色分别检测Runx2、OCN基因的表达。采用SPSS 20.0软件包对数据进行统计学分析。结果hDPSCs经过成骨诱导分化培养后,GLUD1的表达量较对照组明显上升;hDPSCs经shGLUD1病毒转染后,GLUD1表达明显下调;沉默GLUD1表达的hDPSCs体外增殖能力减弱;经成骨诱导分化培养后,与对照组相比,矿化结节形成明显减少;成骨早期标志基因Runx2上调,成骨晚期标志物OCN在mRNA和蛋白水平均显著下调。结论沉默GLUD1表达抑制hDPSCs的体外增殖和矿化形成,影响晚期成骨分化;GLUD1在hDPSCs的成骨分化中具有重要作用。

关键词: GLUD1, 牙髓干细胞, 成骨分化, 增殖, 矿化

Abstract: To investigate the effect of glutamate dehydrogenase 1 (GLUD1) on proliferation, osteogenic differentiation and mineralization of human dental pulp stem cells (hDPSCs). METHODS: hDPSCs were isolated by tissue-explant method in vitro, and shGLUD1 lentivirus was transfected to knock down the expression of GLUD1. RT-PCR and Western blot were performed to detect the expression of GLUD1. CCK8 assay was used to evaluate cell proliferation. After culture with osteogenic inducing medium for 14 days, alizarin red staining was used to detect the formation of mineralization nodules, and RT-PCR and immunofluorescence staining were performed to detect the expression of Runx2 and OCN, respectively. The data were analyzed with SPSS 20.0 software package. RESULTS: The expression of GLUD1 was significantly increased in hDPSCs after osteogenic induction compared with the control. After transfection with shGLUD1 lentivirus, GLUD1 expression was significantly decreased (P<0.05). Compared with the control group, mineralization nodule formation was significantly decreased in shGLUD1 group after osteogenic induction. The expression of OCN (late-staged markers for osteogenic differentiation) were significantly decreased both in mRNA and protein levels, while the expression of Runx2 (early-staged markers for osteoblast differentiation) was up-regulated. CONCLUSIONS: shGLUD1 inhibits the proliferation, mineralization and the late stage of osteogenic differentiation of hDPSCs in vitro. GLUD1 may play an important role in osteogenic differentiation of hDPSCs.

Key words: GLUD1, Human dental pulp stem cells, Osteogenic differentiation, Proliferation, Mineralization

中图分类号:

R781.3

卢皙, 刘上峰, 赵守亮. GLUD1基因对人牙髓干细胞的体外增殖、分化和矿化的影响[J]. 上海口腔医学, 2017, 26(4): 358-362.

LU Xi, LIU Shang-feng, ZHAO Shou-liang. Effect of GLUD1 on proliferation, osteogenic differentiation and mineralization of human dental pulp stem cells[J]. Shanghai Journal of Stomatology, 2017, 26(4): 358-362.

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GLUD1基因对人牙髓干细胞的体外增殖、分化和矿化的影响

Effect of GLUD1 on proliferation, osteogenic differentiation and mineralization of human dental pulp stem cells

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